This invention relates to a glucagon. More particularly, this invention relates to a process for purifying glucagon.
Shortly after the discovery of insulin in 1921 by Banting and Best, several researchers [Murlin, et. al., J. Biol. Chem., 56, 252 (1923) and Kimball and Murlin, J. Biol. Chem., 58, 337 (1924)] noted that a hyperglycemic response was obtained with certain pancreatic extracts of insulin. The factor responsible for the hyperglycemic response was named glucagon. Subsequent research efforts resulted in the purification and crystallization of glucagon; see Staub, et al., Science, 117, 628 (1953 ) and J. Biol. Chem., 214, 619 (1955). Structurally, glucagon is a single polypeptide chain of 29 amino acids. The amino acid sequence of porcine glucagon was established by Bromer, et al., J. Am. Chem. Soc., 79, 2807 (1957).
As already noted, glucagon causes a hyperglycemic response; i.e., an increase in the concentration of glucose in the blood. In this respect, glucagon is in dynamic opposition to insulin which causes a hypoglycemic response, a decrease in the concentration of blood glucose. Consequently, an important use of glucagon is in the treatment of insulin-induced hypoglycemia when hypertonic glucose solution is not available.
Glucagon also has been shown to exhibit a positive inotropic effect; see Farah and Tuttle, J. Pharmacol. Exptl. Therap., 29, 49 (1960). Thus, the administration of glucagon has been shown to produce an increase in the contractal force of the heart. This has led to an extensive use of glucagon in the treatment of hypodynamic heart disorders in which an increase in cardiac contractile force is required [Van der Ark, et. al., Amer. Heart J., 79, 481 (1970)].
Additionally, it has been recognized that glucagon exhibits various other kinds of biological activity. For example, glucagon has been used to relax the duodenum for X-ray visualization in hypotonic duodenography [Miller, et al., Radiology, 108, 35 (1973)]. Glucagon also is active as a diuretic, as a bronchodilator, in reducing gastric secretion, and in reducing the level of blood lipids and blood cholesterol. Finally, glucagon has been used in the treatment of pancreatitis [Stremmel, Pharmakotherapie In Kurze, 116, 69 (1974)].
The above-described recognized practical uses of glucagon are placing increased demands on the availability and purity of glucagon.
It is well known that both insulin and glucagon are produced within the pancreas. While the isolation and purification of insulin have reached a high degree of sophistication, prior art procedures for isolating and purifying glucagon still present various problems, particularly with respect to purity, glucagon degradation, and recovery efficiency. Such problems at one time were due in part to the close relationship between insulin and glucagon. Improvements in insulin purification procedures, however, have eliminated or minimized the contribution to such problems from insulin processing procedures.
At the present time, such problems are associated primarily with the prior art procedures for purifying glucagon. For example, fibril formation is carried out at a low pH, typically about pH 2.0, an environment which contributes to increased glucagon hydrolysis. The products of hydrolysis in general exhibit reduced activity. For example, desamido glucagon possesses only about 60 percent of the hormonal activity of glucagon. In addition, fibril formation is dependent upon glucagon purity; as purity decreases, fibril formation becomes increasingly difficult or even impossible.
Because glucagon has a tendency to gel and to form fibrils in acidic solutions, chromatographic procedures involving acidic glucagon solutions tend to experience glucagon losses from the gelation and precipitation of glucagon in the column. Furthermore, glucagon tends to aggregate in acidic solutions, thereby significantly reducing the selectivity and effectiveness of gel filtration (gel exclusion chromatography) under acidic conditions.